Selenium, an essential trace element, is incorporated into proteins as selenocysteine (Sec). There are twenty-five selenoproteins in humans, which play a variety of critical roles in health and disease. The proper expression of the selenoproteome is important for the normal function of the cardiovascular, endocrine, immune, muscular, and nervous systems. Sec is encoded by UGA, which normally signals the termination of protein synthesis. The recoding of UGA as Sec during translation depends on the Sec Insertion Sequence (SECIS) element in the 3' UTR of the selenoprotein mRNA. In the absence of a SECIS, the ribosome terminates at the UGA and produces a truncated protein, which lacks Sec. Substantial progress has been made in elucidating the role of the SECIS in dictating the expression of the selenoproteome. However, much remains to be learned about how the efficiency of Sec incorporation is regulated and how the ribosome discriminates between the UGA/Sec codon and the authentic stop codon. These central questions are particularly critical for understanding the biosynthesis of Selenoprotein S (SelS) and other selenoproteins that contain a C-terminal Sec residue, as the UGA/Sec codon is only a few nucleotides upstream of the stop codon. Our results suggest that the production of full-length SelS and the truncated protein that lacks Sec is highly regulated. We identified two sequences outside of the SECIS element in the SelS 3' UTR that either promote or inhibit Sec incorporation. Both of these regions interact with unknown proteins. We also provide evidence that SelS and other selenoprotein mRNAs contain the modified nucleotide pseudouridine (?) in vivo. The central hypothesis of this proposal is that 3' UTR sequences outside of the SECIS and the modified nucleotide ? are part of the cis-acting code in selenoprotein mRNAs that regulates the efficiency of Sec incorporation. In this proposal, we will fully define the role of the proximal stem-loop in the SelS 3' UTR in promoting Sec insertion and test the hypothesis that other selenoproteins, which contain a C-terminal Sec are regulated by a similar mechanism. We will identify the sequence in the distal part of the SelS 3'UTR that inhibits Sec insertion as well as the proteins that bind to this region to mediate repression. Finally, we will define the ? landscape of the selenoprotein transcriptome and test the hypothesis that Sec may be encoded by ?GA in endogenous selenoprotein mRNAs. We expect that our studies will identify novel determinants that regulate the expression of the selenoproteome. Our research will also have a broad impact by providing new insight into the role of mRNA modifications in mammalian cells.